Currently, there is no effective vaccine for HIV infection. HIV vaccines can potentially be used for prevention of infection or therapeutically to control the level of HIV replication post-exposure. The overall goal of this 5-year research proposal is to develop nanoparticle-based HIV-1 vaccines to elicit enhanced Th1, cytotoxic T lymphocyte (CTL), and humoral immune responses to recombinant Tat (1-72) and Gag p24 proteins. The immunogenicity of the developed nanoparticle-based HIV-1 Tat (1-72) and Gag p24 vaccine will further be enhanced by, 1) the attachment of a dendritic cell targeting ligand, mannopentaose, to the nanoparticles, and 2) coating the nanoparticles with CpG to target the toll-like receptor-9 (TLR-9). Optimized nanoparticles vaccines will be compared to a heterologous prime boost strategy based on vaccinia expressing antigen and adjuvanted protein. Recombinant HIV Tat (1-72) and Gag p24 proteins and his-tagged proteins will be synthesized and purified to homogeneity by Dr. Nath's laboratory at Johns Hopkins and tested for bioactivity. In Dr. Mumper's laboratory at the University of Kentucky, the proteins will be either coated on anionic nanoparticles (Type 1) or attached to nanoparticles made with a small amount of accessible nickel at the surface (Type 2). Both types of solid nanoparticles (<100 nm) will be made from novel oil-in-water microemulsion precursors. Dr. Mumper's lab will characterize the nanoparticles, perform in-vitro uptake and activation studies in mouse and human dendritic cells, and perform animal experiments to determine the humoral immune responses of the nanoparticle-based formulations, and perform immunohistochemical analyses. Dr. Woodward's laboratory at the University of Kentucky will be responsible for ELISPOT, CTL, multi-probe ribonuclease, tetramer, cell proliferation assays, as well as confocal microscopy and flow cytometry experiments. Dr. Nath's laboratory will perform studies demonstrating that sera from immunized mice can suppress Tat-induced LTR-transactivation, and will assess the effect of Tat and Gag p24 antisera on HIV replication. In addition, Dr. Nath's lab will produce vaccinia expressing antigen.